Biochemical characterization of autolysin from pathogenic type 19F and the non-pathogenic strain R61 was studied. The enzymatic activities of autolysin in relation to the electrophoretic pattern and biological function were examined. Pheumococcal autolysin exhibited the hydrolytic activity to remove ribose, glucosamine, galactosamine and lysine from the cell wall, indicating that it contained glucosidases and amidases, mainly N-acetylmuramic acid-L-Alanine amidase and N-acetylmuramidase.The autolysin was purified from pneumococcal cells by the methods of sonic disruption with sodium deoxycholate, ammonium sulfate precipitation, bio- gel P-4, DEAE-cellulose, and hydroxyapatite chromatography. The electrophoretic pattern of a purified autolysin, amidase, exhibited a single band with 36 KDa molecular weight. The yield was approximately 0.1% of the crude protein extract. The specific enzymatic activity increased 150 fold. Type 19F and R61 autolysin stimulated the release of pneumolysin from the cells and increased the pneumolysin activity 4-8 fold in type 19F cells.